As an analyzing method applicable for such a three-dimensional analyzing device, there is known a fluorescence correlation method as disclosed, for example, in a non-patent document: “Fluorescence Correlation Spectroscopy in DNA Analysis”, authored by Masataka KINJO, Journal of The Japan Society for Precision Engineering, Vol. 65, No. 2, 1999, pp. 175-180. The fluorescence correlation method has long been used in analysis on a diffusive motion such as Brownian movement of particles, in which, as shown in a principle diagram of FIG. 13, physical values associated with the size or number of fluorescence molecules are analyzed based on a fluorescence correlation function of the amplitude and duration of fluctuation, which is obtained by irradiating a narrow laser beam as an exciting beam to a dilute solution of fluorescence molecules, and measuring a fluorescence intensity in an observation region exposed to the laser beam for a long time. Since the fluorescence intensity is proportional to the number N of fluorescence molecules included in the observation region, the intensity of fluctuation in terms of S/N can be expressed as (1/N)1/2.
In such a fluorescence correlation method, the correlation time τ0, i.e., the length of time during which the fluorescence correlation function as a physical value decreases by half, can be expressed as the following formula (1):
                    τ0        =                              W            2                                4            ⁢            D                                              (        1        )            where D is a translational diffusion coefficient of the fluorescence molecule, and W is a beam radius of the laser beam when the intensity distribution function thereof in its radial direction follows the Gaussian distribution. In a physical sense, the correlation time τ0 corresponds to the length of time during which fluorescence molecules pass across the laser beam by diffusion.
In the fluorescence correlation method, the fluorescence fluctuation is generally measured with an output current f(t) of a photoelectron multiplier that receives the fluorescence, wherein the output current f(t) is proportional to the fluorescence quantity when the radius of the laser beam is not extremely large. The fluorescence correlation function is equivalent a correlation function G(τ) of the output current f(t) with respect to time. The fluorescence correlation function G(τ) can be expressed as the following formula (2), which can be simplified as the following formula (3) when the laser beam intensity substantially follows the Gaussian distribution.
                              G          ⁡                      (            τ            )                          =                                            ∫              0              ∞                        ⁢                                          f                ⁡                                  (                  t                  )                                            ⁢                              f                ⁡                                  (                                      t                    +                    τ                                    )                                            ⁢                                                          ⁢                              ⅆ                t                                                                        ∫              0              ∞                        ⁢                                          f                ⁡                                  (                  t                  )                                            ⁢                              f                ⁡                                  (                  t                  )                                            ⁢                                                          ⁢                              ⅆ                t                                                                        (        2        )                                          G          ⁡                      (            τ            )                          =                              1            N                    ·                      1                          1              +                              τ                /                                  τ                  0                                                                                        (        3        )            
As explained above, the fluorescence correlation method makes it possible to measure, basically in the same principle, any physical value from which a translational diffusion coefficient can be obtained, provided that the physical value is a thermodynamic value that gives a fluorescence fluctuation. For example, a fluorescence fluctuation can be observed when fluorescence molecules pass across the laser beam by flowage thereof. If a fluorescence molecule is bound with another molecule in a chemical reaction, for example, a molecule velocity can be observed as a fluctuation. In other words, the development of the chemical reaction can be known in a real time manner. In addition, a rotational movement of a molecule can also be measured with ellipsometry.
Further, the number of molecules included in the observation region can be measured directly, based on the intensity of the fluorescence correlation function G(τ). More specifically, a fluctuation f(t) during a certain measuring time long enough for an expected fluctuation to be completed is measured, which is then used for obtaining a correlation function with the formula (2). Generally, a CW (continuous wave) argon laser or krypton laser is used as an exciting beam source for analyzing a fluorescence correlation of a pigment molecule. A representative system for the fluorescence correlation analysis used in the prior art is shown in FIG. 15.
In the system shown in FIG. 15, an argon laser 51 is used as an exciting beam source, from which a laser beam is emitted and transmitted through a beam splitter 52, to be collected by a lens 53 and irradiated to a specimen solution 54 containing fluorescence molecules. The fluorescence in the specimen solution 54 is collimated by the lens 53 and reflected by the beam splitter 52, to be collected by a lens 55. The collected fluorescence passes through a pinhole 56 to be received by a detector 57, such as a photoelectron multiplier or CCD. The output of the detector 57 is amplified by a preamplifier 58, converted by an analog/digital (A/D) converter 59 into digital data, and then inputted to an operational equipment 60 comprising a computer etc., for calculating the correlation function G(τ).
The system of the type shown in FIG. 15 is also disclosed in the non-patent document identified above.
According to various experimental studies conducted by the inventors, however, it has been found from practical viewpoint that the above-mentioned system for a fluorescence correlation analysis as used in the prior art is still to be improved in the following points.
As the fluorescence correlation method is based on detection of fluctuation, the number of the fluorescence molecules is preferably as small as possible, particularly one molecule if possible. However, the region exposed to a beam inducing a fluorescence has a lower limit in size, i.e., a diffraction limit that is defined by the numerical aperture (NA) of the lens 53 and the wavelength λ of the beam, as expressed by the following formula (4). Thus, as the absolute quantity of fluorescence molecules increases, the region exposed to the beam should be narrowed down correspondingly, to reduce the number of fluorescence molecules passing across the observation region.
                    W        =                  1.22          ⁢                      λ            NA                                              (        4        )            
Therefore, even when the lens 53 comprises an immersion lens of NA=1.4 and a laser beam of λ=500 nm is used as the exciting beam in FIG. 15, for example, a focusing radius W of the laser beam is 436 nm at the lowest. Moreover, the size in the depth direction of the beam coincides with the very thickness of the specimen solution 54. Thus, in carrying out a measurement in practice, it is required to extremely lessen the density of fluorescence molecules contained in the specimen solution 54 to be analyzed, which is a significant obstacle to the practical utility.
In order to limit the size of the observation region in the depth direction of the beam, a pinhole 56 is generally provided at the confocal position for cutting the fluorescence emitted from a region outside the focal plane. However, even with the provision of such a pinhole, the resolution in the depth direction nevertheless remains on the order of several micrometers. Further, the positioning of the pinhole 56 is delicate, with the result that the fluorescence to be observed is also cut in many instances.
For the reasons explained above, it has been difficult to apply the fluorescence correlation method to a concentrated solution, besides that a high three-dimensional spatial resolution cannot be expected, either.